The interaction between FUra/LV and recombinant human IFN-gamma is synergistic in human colon carcinoma cells, dependent on Fura/LV-induced DNA damage, the Fas signaling pathway, and independent of p53. Preliminary studies also demonstrate sites of interaction in addition to Fas that involve caspases, the IAP protein survivin, and p21Cip1. A Phase I trial of FUra/LV combined with IFN-gamma has demonstrated responses in heavily pretreated and untreated patients. A Phase II trial will now be conducted in patients with previously untreated or previously treated metastatic colorectal cancer, with the overall goal to elucidate the prognostic significance of expression of genes that influence FUra/LV, Fas and IFN-gamma signaling and the synergistic interaction between these pathways in the induction of apoptosis, cellular cytotoxicity and response. We will elucidate, by tissue microarray (protein) or Real Time PCR (mRNA), the prognostic significance of genes known to determine sensitivity to FUra/LV (TS,DPD, TP, Cox-2) or influence the Fas or IFN-gamma signaling pathways (Bcl-2, Bax, p53, p21Cip1, Fas, caspases -1, -3, -8, survivin, STAT1) in colon carcinoma cells. Gene expression will be determined in metastases both before (sample 2) and at 4 days after (sample 3) FUra/LV/IFN-gamma treatment, and in primary tumors (sample 1) from the same patients. The significance of changes in gene expression in metastases in predicting time to disease progression, response, and how changes in Fas expression correlate with elevated expression in CD15+ and CD56+ cells, as well as toxicity and IFN-gamma pharmacokinetics, will be determined. Using oligonucleotide microarray analyses (Affymetrix), the hypothesis is that we will elucidate additional genes that are independent molecular determinants for predicting time to disease progression, response at 8 weeks, overall response, intrinsic drug resistance or acquired drug resistance following FUra/LV/IFN-gamma therapy. It is anticipated that the proposed studies will identify critical targets that 1) are convergent between death receptor, FUra/LV and IFN-gamma signaling pathways, 2) determine synergistic interactions between FUra/LV and IFN-gamma in metastatic colorectal carcinoma, 3) predict for response, intrinsic drug resistance or acquired drug resistance in FUra/LV/IFN-gamma therapy, and 4) will further our knowledge in the identification of new targets for therapeutic intervention, and in the development of highly effective therapeutic approaches in the treatment of this disease, based upon rational preclinical design.